Oligonucleotides have the potential to recognize unique sequences of DNA or RNA with a remarkable degree of specificity. For this reason they have been considered as promising candidates to realize gene specific therapies for the treatment of malignant, viral and inflammatory diseases. Two major strategies of oligonucleotide-mediated therapeutic intervention have been developed, namely, the antisense and antigene approaches. The antisense strategy aims to down-regulate expression of a specific gene by hybridization of the oligonucleotide to the specific mRNA, resulting in inhibition of translation. Gewirtz et al. (1998) Blood 92, 712-736; Crooke (1998) Antisense Nucleic Acid Drug Dev. 8, 115-122; Branch (1998) Trends Biochem. Sci. 23, 45-50; Agrawal et al. (1998) Antisense Nucleic Acid Drug Dev. 8, 135-139. The antigene strategy proposes to inhibit transcription of a target gene by means of triple helix formation between the oligonucleotide and specific sequences in the double-stranded genomic DNA. Helene et al. (1997) Ciba Found. Symp. 209, 94-102. Clinical trials based on the antisense approach are now showing that oligonucleotides can be administered in a clinically relevant way and have few toxic side effects. Gewirtz et al. (1998) Blood 92, 712-736; Agrawal et al. (1998) Antisense Nucleic Acid Drug Dev. 8, 135-139.
Whereas both the antisense and antigene strategies have met with some success, it has become clear in recent years that the interactions of oligonucleotides with the components of a living organism go far beyond sequence-specific hybridization with the target nucleic acid. Recent studies and reexamination of early antisense data have suggested that some of the observed biological effects of antisense oligonucleotides cannot be due entirely to Watson-Crick hybridization with the target mRNA. In some cases, the expected biological effect (e.g. inhibition of cell growth or apoptosis) was achieved, but this was not accompanied by a down regulation of the target protein and was thus unlikely to be a true antisense effect. White et al. (1996) Biochem. Biophys. Res. Commun. 227, 118-124; Dryden et al. (1998) J. Endocrinol. 157, 169-175. In many cases, it was demonstrated that other non-sequence specific oligonucleotides could exert biological effects that equaled or exceeded the antisense sequence. Barton et al. (1995) Br. J. Cancer 71, 429-437; Burgess et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4051-4055; Benimetskaya et al. (1997) Nucleic Acids Res. 25, 2648-2656. Though there is currently a high awareness among antisense investigators of the importance of appropriate control oligonucleotides, and the necessity of demonstrating inhibition of target protein production (Stein (1998) Antisense Nucleic Acid Drug Dev. 6, 129-132), the mechanism of non-antisense effects is poorly understood.
In particular, phosphodiester and phosphorothioate oligodeoxynucleotides containing contiguous guanosines (G) have been repeatedly found to have non-antisense effects on the growth of cells in culture. Burgess et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4051-4055; Benimetskaya et al. (1997) Nucleic Acids Res. 25, 2648-2656; Saijo et al. (1997) Jpn. J. Cancer Res. 88, 26-33. There is evidence that this activity is related to the ability of these oligonucleotides to form stable structures involving intramolecular or intermolecular G-quartets. Burgess et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4051-4055; Benimetskaya et al. (1997) Nucleic Acids Res. 25, 2648-2656. These are square planar arrangements of four hydrogen-bonded guanines that are stabilized by monovalent cations. Such structures are thought to play an important role in vivo an 4 putative quartet forming sequences have been identified in telomeric DNA (Sundquist et al. (1989) Nature 342, 825-829), immunoglobulin switch region sequences (Sen et al. (1988) Nature 334, 364-366), HIV1 RNA (Sundquist et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3393-3397), the fragile X repeat sequences (Fry et al (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4950-4954) and the retinoblastoma gene (Murchie et al. (1992) Nucleic Acids Res. 20, 49-53).
It has been suggested that non-antisense effects may be due to sequestration of intracellular or surface proteins by the oligonucleotide. Gold et al. (1995) Annu. Rev. Biochem. 64, 763-797; Stein (1997) Ciba Found. Symp. 209, 79-89. For G-rich oligonucleotides that can form folded, G-quartet containing structures, this binding is thought to be mediated not by recognition of the primary sequence of the oligonucleotides, but rather of their unique three-dimensional shapes. However, the protein targets of these oligonucleotides have not been well characterized.
Oligonucleotides are polyanionic species that are internalized in cells, probably by receptor-mediated endocytosis. Vlassov et al. (1994) Biochim. Biophys. Acta 1197, 95-108. They are likely to interact with many biomolecules within the cell and also in the extracellular membrane by virtue of both their charge and their shape, as well as sequence-specific interactions. The proteins that bind to oligonucleotides and mediate non-antisense effects have not yet been unequivocally identified.
The present application identifies a G-rich oligonucleotide binding protein, and the ability of a G-rich oligonucleotide to bind to this protein is correlated with its propensity to form G-quartets, and with its ability to inhibit the growth of tumor cells.
Applicants have described G-rich oligonucleotides (GROs) that have potent growth inhibitory effects that are unrelated to any expected antisense or antigene activity. While the mechanism of these effects has not yet been specifically delineated, Applicants have demonstrated that the antiproliferative effects of these oligonucleotides are related to their ability to bind to a specific cellular protein. Because the GRO binding protein is also recognized by anti-nucleolin antibodies, Applicants have concluded that this protein is either nucleolin itself, or a protein of a similar size that shares immunogenic similarities with nucleolin.
Nucleolin is an abundant multifunctional 110 kDa phosphoprotein thought to be located predominantly in the nucleolus of proliferating cells (for reviews, see Tuteja et al. (1998) Crit. Rev. Biochem. Mol. Biol. 33, 407-436; Ginisty et al. (1999) J Cell Sci. 112, 761-772). Nucleolin has been implicated in many aspects of ribosome biogenesis including the control of rDNA transcription, pre-ribosome packaging and organization of nucleolar chromatin. Tuteja et al. (1998) Crit. Rev. Biochem. Mol. Biol. 33, 407-436; Ginisty et al. (1999) J Cell Sci. 112, 761-772; Ginisty et al. (1998) EMBO J. 17, 1476-1486. Another emerging role for nucleolin is as a shuttle protein that transports viral and cellular proteins between the cytoplasm and nucleus/nucleolus of the cell. Kibbey et al. (1995) J. Neurosci. Res. 42, 314-322; Lee et al. (1998) J. Biol. Chem. 273, 7650-7656; Waggoner et al. (1998) J. Virol. 72, 6699-6709. Nucleolin is also implicated, directly or indirectly, in other roles including nuclear matrix structure (Gotzmann et al. (1997) Electrophoresis 18, 2645-2653), cytokinesis and nuclear division (Leger-Silvestre et al. (1997) Chromosoma 105, 542-52), and as an RNA and DNA helicase (Tuteja et al. (1995) Gene 160, 143-148). The multifunctional nature of nucleolin is reflected in its multidomain structure consisting of a histone-like N-terminus, a central domain containing RNA recognition motifs, and a glycine/arginine rich C-terminus. Lapeyre et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1472-1476. Levels of nucleolin are known to relate to the rate of cellular proliferation (Derenzini et al. (1995) Lab. Invest. 73, 497-502; Roussel et al. (1994) Exp. Cell Res. 214, 465-472.), being elevated in rapidly proliferating cells, such as malignant cells, and lower in more slowly dividing cells. For this reason, nucleolin is an attractive therapeutic target.
Although considered a predominantly nucleolar protein, the finding that nucleolin was present in the plasma membrane is consistent with several reports identifying cell surface nucleolin and suggesting its role as a cell surface receptor. Larrucea et al. (1998) J. Biol. Chem. 273, 31718-31725; Callebout et al. (1998) J. Biol. Chem. 273, 21988-21997; Semenkovich et al. (1990) Biochemistry 29, 9708; Jordan et al. (1994) Biochemistry 33, 14696-14706.
Previously, several mechanisms were proposed to explain the non-sequence-specific effects of oligonucleotides. These included binding to cellular receptors (Rockwell et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 6523-6528; Coulson et al. (1996) Mol. Pharmacol. 50, 314-325), modulation of cytokine or growth factor activity (Hartmann et al. (1996) Mol. Med. 2, 429-438; Sonehara et al. (1996) J Interferon Cytokine Res. 16, 799-803; Fennewald et al. (1995) J. Biol. Chem. 270, 21718-21721; Guvakova et al. (1995) J. Biol. Chem. 270, 2620-2627; Scaggiante et al. (1998) Eur. J. Biochem. 252, 207-215), inhibition of cell cycle progression (Burgess et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4051-4055), changes in cell adhesion (Saijo et al. (1997) Jpn. J. Cancer Res. 88, 26-33) and binding to an uncharacterized 45 kDa protein (Ramanathan et al. (1994) J. Biol. Chem. 269, 24564-24574). The immunostimulatory properties of oligonucleotides containing 5′-CG-3′ sequences have also been described (McCluskie et al. (1998) J. Immunol. 161, 4463-4466), but it seems unlikely that they are related to the effects Applicants have observed.
In this present application, Applicants have identified an oligonucleotide binding protein and shown a correlation between binding to this protein and antiproliferative activity for a series of G-rich oligonucleotides. These findings are strongly suggestive of a mechanistic role for this protein in non-antisense oligonucleotide-mediated inhibition of cell growth. The basis for recognition of GROs by nucleolin is not obvious from the sequences of the oligonucleotides tested, but may relate to their propensity to form particular G-quartet structures.
The relationship between nucleolin binding and antiproliferative activity for other, non-G-rich, oligonucleotides has not yet been fully evaluated. One mixed sequence oligonucleotide (MIX1) was found to bind nucleolin, although it had no growth inhibitory effect. Nucleolin contains RNA binding domains that can recognize specific sequences of RNA or single-stranded DNA. Dickinson et al. (1995) Mol. Cell. Biol. 15, 456-465; Ghisolfi et al. (1996) J. Mol. Biol. 260, 34-53. It is possible that this particular oligonucleotide contains a sequence or structure that resembles such a recognition element.
In support of the Applicants' findings that nucleolin binds to G-rich oligonucleotides, recent reports have demonstrated that nucleolin can bind to other G-quartet forming sequences, such as immunoglobulin switch regions and ribosomal gene sequences (Dempsey et al. (1999) J. Biol. Chem. 274, 1066-1071 and Hanakai et al. (1999) J. Biol. Chem. 274, 15903-15912). It is possible that nucleolin has currently undefined functions in vivo that depend on recognition of G-rich sequences in, for example, ribosomal DNA switch region sequences or telomeres.
The synthesis of nucleolin is positively correlated with increased rates of cell division, and nucleolin levels are therefore higher in tumor cells as compared to most normal cells. In fact, nucleolin is one of the nuclear organizer region (NOR) proteins whose levels, as measured by silver staining, are assessed by pathologists as a marker of cell proliferation and an indicator of malignancy. Nucleolin is thus a tumor-selective target for therapeutic intervention, and strategies to reduce the levels of functional nucleolin are expected to inhibit tumor cell growth.
The consequences of nucleolin inhibition on the growth of cells have not been well studied, but inhibition of a protein whose functions include ribosome production, nuclear transport and cell entry should have profound effects on the growth of cells.